| 主权项 |
1. A method of treating a biomass comprising cellulose, the method comprising contacting the biomass with a host cell that expresses a recombinant chimeric polypeptide comprising at least two segments from at least two different parental cellobiohydrolase I (CBH I) polypeptides, wherein the chimeric polypeptide comprises from N- to C- terminus:(segment 1)-(segment 2)-(segment 3)-(segment 4)-(segment 5)-(segment 6)-(segment 7)-(segment 8);
wherein:
segment 1 comprises a sequence that is at least 98-100% identical to amino acid residue from 1 or from 18 or 19 to x1 of SEQ ID NO:2 (“1”), SEQ ID NO:4 (“2”), SEQ ID NO:6 (“3”), SEQ ID NO:8 (“4”), or SEQ ID NO:10 (“5”);segment 2 comprises a sequence that is at least 98-100% identical to amino acid residue x1 to x2 of SEQ ID NO:2 (“1”), SEQ ID NO:4 (“2”), SEQ ID NO:6 (“3”), SEQ ID NO:8 (“4”), or SEQ ID NO:10 (“5”);segment 3 comprises a sequence that is at least 98-100% identical to amino acid residue x2 to x3 of SEQ ID NO:2 (“1”), SEQ ID NO:4(“2”), SEQ ID NO:6 (“3”), SEQ ID NO:8 (“4”), or SEQ ID NO:10 (“5”);segment 4 comprises a sequence that is at least 98-100% identical to amino acid residue x3 to x4 of SEQ ID NO:2 (“1”), SEQ ID NO:4(“2”), SEQ ID NO:6 (“3”), SEQ ID NO:8 (“4”), or SEQ ID NO:10 (“5”);segment 5 comprises a sequence that is at least 98-100% identical to amino acid residue x4 to x5 of SEQ ID NO:2 (“1”), SEQ ID NO:4 (“2”), SEQ ID NO:6 (“3”), SEQ ID NO:8 (“4”), or SEQ ID NO:10 (“5”);segment 6 comprises a sequence that is at least 98-100% identical to amino acid residue x5 to x6 of SEQ ID NO:2 (“1”), SEQ ID NO:4(“2”), SEQ ID NO:6 (“3”), SEQ ID NO:8 (“4”), or SEQ ID NO:10 (“5”);segment 7 comprises a sequence that is at least 98-100% identical to amino acid residue x6 to x7 of SEQ ID NO:2 (“1”), SEQ ID NO:4(“2”), SEQ ID NO:6 (“3”), SEQ ID NO:8 (“4”), or SEQ ID NO:10 (“5”); andsegment 8 comprises a sequence that is at least 98-100% identical to amino acid residue x7 to x8 of SEQ ID NO:2 (“1”), SEQ ID NO:4(“2”), SEQ ID NO:6 (“3”), SEQ ID NO:8 (“4”), or SEQ ID NO:10 (“5”); wherein x1 is residue 47, 48, 49, 50, 51, or 52 of SEQ ID NO:2, 4, 6, or 8, or residue 48, 49, 50, 51, 52 or 53 of SEQ ID NO:10; x2 is residue 92, 93, 94 95, 96 or 97 of SEQ ID NO:2 or 10, or residue 91, 92, 93, 94, 95, or 96 of SEQ ID NO:4, 6, or 8; x3 is residue 127, 128, 129, 130, 131 or 132 of SEQ ID NO:2, or residue 125, 126, 127, 128, 129 or 130 of SEQ ID NO:4 or 6, or residue 126, 127, 128, 129, 130 or 131 of SEQ ID NO:8, or residue 123, 124, 125, 126, 127 or 128 of SEQ ID NO:10; x4 is residue 175, 176, 177, 178, 180 or 181 of SEQ ID NO:2, or residue 173, 174, 175, 176, 177 or 178 of SEQ ID NO:4 or SEQ ID NO:6, or residue 174, 175, 176, 177, 178 or 179 of SEQ ID NO:8, or 171, 172, 173, 174, 175, or 176 of SEQ ID NO:10; x5 is 221, 222, 223, 224, 225, or 226 of SEQ ID NO:2, or residue 219, 220, 221, 222, 223 or 224 of SEQ ID NO:4 or SEQ ID NO:6, or residue 220, 221, 222, 223, 224 or 225 of SEQ ID NO:8, or 217, 218, 219, 220, 221 or 222 of SEQ ID NO:10; x6 is residue 268, 269, 270, 271, 272 or 273 of SEQ ID NO:2, or residue 266, 267, 268, 269, 270 or 271 of SEQ ID NO:4 or SEQ ID NO:6, or residue 267, 268, 269, 270, 271 or 272 of SEQ ID NO:8, or 264, 265, 266, 267, 268 or 269 of SEQ ID NO:10; x7 is residue 384, 385, 386, 387, 388 or 389 of SEQ ID NO:2, or residue 385, 386, 387, 388, 389 or 390 of SEQ ID NO:4, or residue 378, 379, 380, 381, 382 or 383 of SEQ ID NO:6, or residue 383, 384, 385, 386, 387 or 388 of SEQ ID NO:8 or 10; and x8 is an amino acid residue corresponding to residue 454 of SEQ ID NO:2, residue 457 of SEQ ID NO:4, residue 458 of SEQ ID NO:6, residue 453 of SEQ ID NO:8, residue 455 of SEQ ID NO:10 or the C-terminus of the polypeptide having the sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10, wherein the chimeric polypeptide is about 437 or 441 amino acid residues in length, and wherein the chimeric polypeptide has cellobiohydrolase activity and improved thermostability, pH stability and/or expression compared to a CBH I polypeptide comprising SEQ ID NO:2, 4, 6, 8 or 10. |
